University of Groningen
Characterization and computation-supported engineering of an ω-transaminase Meng, Qinglong
DOI:
10.33612/diss.172243517
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Publication date: 2021
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Meng, Q. (2021). Characterization and computation-supported engineering of an ω-transaminase. University of Groningen. https://doi.org/10.33612/diss.172243517
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CHAPTER Ⅳ
Asymmetric synthesis of optically pure aliphatic
amines with an engineered robust ω-transaminase
Linhan Dong, Qinglong Meng, Carlos Ramírez-Palacios, Hein J. Wijma, Siewert J. Marrink and Dick B. Janssen96
Abstract
The production of chiral amines by transaminase-catalyzed amination of ketones is an important application of biocatalysis in synthetic chemistry. It requires transaminases that show high enantioselectivity in asymmetric conversion of the ketone precursors. A robust derivative of an ω-transaminase from Pseudomonas jessenii (PjTA-R6) which naturally acts on aliphatic substrates was constructed previously in our group. Here, we explore the catalytic potential of this
thermostable enzyme for the synthesis of optically pure aliphatic amines and compare it to the well-studied transaminases from Vibrio fluvialis (VfTA) and Chromobacterium violaceum (CvTA). The product yields indicated improved performance of PjTA-R6 over the other transaminases and in most cases the optical purity of the produced amine was above 99% ee. Structural analysis revealed that the substrate binding poses are influenced and restricted by the switching arginine and that this accounts for differences in substrate specificities. Rosetta docking calculations with
external aldimine structures showed a correlation between docking scores and synthetic yields. The results show that PjTA-R6 is a promising biocatalyst for the asymmetric synthesis of aliphatic amines with a product spectrum that can be explained by its structural features.
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Introduction
Optically pure amines are of great interest for the pharmaceutical and wider fine chemicals industry as they can serve as intermediates in the preparation of a diversity of biologically active compounds1−3. For example, chiral amines are used as building blocks in the synthesis of pharmaceuticals used in the treatment of Alzheimer's disease4, malaria5, or as antitumor agents6. The enzymatic production of such amines is widely studied for furthering the application of biocatalysis in environmentally benign synthetic processes7,8.
Transaminases (TAs) catalyze the transfer of amino groups from amines (amino donors) to aldehydes or ketones (amino acceptors), with pyridoxal-5′ phosphate (PLP) as the cofactor
(Scheme 1)2,9. Whereas α-transaminases act on substrates bearing a carboxylate group flanking the carbon carrying the amine functionality that is exchanged, ω-transaminases (ω-TAs) act on non-proteinogenic amino acids with the amine and carboxylate groups at terminal positions. Many ω-TAs and related enzymes are called amine transaminases (ATA) as they act on substrates which lack a carboxylate functionality. This relaxed substrate specificity allows amination of various ketones when added as amino acceptor10. Mechanistically, in the first half-reaction the amino donor aminates the PLP to form in several proton transfer steps a pyridoxamine intermediate (PMP). In the second half-reaction the ketone acceptor binds to the PMP enzyme to form the aminated product and regenerate PLP (Scheme 1). Recent efforts to tailor the ω-TA substrate scope have focused on stabilization of the Michaelis complex of the ketone substrate11−14 with little or no attention to reaction intermediates. The external aldimine is a key intermediate in the
transamination reaction. Depending on the half reaction under consideration, the external aldimine can either undergo proton abstraction by the catalytic lysine to form a quinonoid intermediate, or abstract a proton from lysine to form a geminal diamine intermediate15. Both formation and conversion of the external aldimine go through the formation of high-energy transition state structures, and their corresponding reactions are rate-limiting. Therefore, we hypothesized that substrates capable of forming more stable external aldimine complexes would overall have better reactivities.
Prior work on the catalytic abilities of ω-TAs indicates versatile substrate spectra and (S)- or (R)-enantioenrichment of amines formed by conversion of prochiral ketones16. Compared with traditional chemical synthesis, harsh conditions and heavy metal catalysts are avoided by
enzymatic reactions, displaying the green aspects of biocatalysis3,7. Moreover, the use of cheap amine donors or regeneration of the amine donor in TA-catalyzed amination enables one-pot asymmetric synthesis3,17. Therefore, the production of optically pure amines applying ω-TAs is considered promising from an application point of view. A large variety of transaminases that are active in the enzymatic synthesis of aromatic amines have been described16.
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Scheme 1. ω-Transaminase-catalyzed transamination with isopropylamine as the amino donor. The aldehyde/ketone substrate will bind to the PMP-enzyme to form an external aldimine. The relatively large (RL) or small (RS) ketone/amine rest groups are accommodated in the large or small binding pockets of the
transaminase. The excess of isopropylamine drives the equilibrium towards the production of the target amine.
In comparison, biocatalytic synthesis of chiral aliphatic amines is not well-studied and reports on asymmetric synthesis of aliphatic amines by TAs are scarce16. Nonetheless, aliphatic amines are frequently seen as important intermediates of pharmaceuticals and other biologically active compounds7,18. Thus, transaminases capable of producing various enantiopure aliphatic amines would diversify and strengthen the biocatalytic toolbox.
Apart from the need for an active enzyme for aliphatic amine synthesis, there are also several technical challenges concerning TA-catalyzed amine production. First, the reaction equilibrium needs to be shifted from the amino donor towards the production of the target amines. Often, this is achieved by using an excess of the amino donor, sometimes accompanied by the removal of the side-product7,16,19,20. Second, to use and fully dissolve a high concentration of the ketone substrate that should undergo amination, a cosolvent is sometimes added1,21. Besides overcoming equilibrium issues, the reaction rate may need to be accelerated by using a high temperature. The availability of a robust ω-TA is desired in such scenarios of high amino donor concentrations, presence of cosolvents, and high reaction temperatures3.
The discovery22 and characterization23 of a class III (S)-selective ω-TA from Pseudomonas jessenii (PjTA) were described previously. Identified as a key enzyme in the caprolactam
degradation pathway of P. jessenii, PjTA converts aminohexanoic acid (AHA) to
6-oxohexanoic acid (6-OHA). Our group has deepened the understanding of this enzyme by solving crystal structures and measuring its activities using over 40 different amine substrates23. A robust
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six-fold mutant of PjTA (PjTA-R6) was recently constructed using a framework for rapid enzyme stabilization by computational library design (FRESCO)24. The natural substrate of PjTA is a linear aliphatic ω-amino acid, which suggests PjTA may have potential for the synthesis of aliphatic amines. Moreover, the robust mutant PjTA-R6 is expected to tolerate a high concentration of amino donor, the presence of cosolvent, and an enhanced reaction temperature24. Thus, in the work reported here, we explored PjTA-R6 for the synthesis of aliphatic amines for which the wild-type PjTA was not considered as attractive due to its lower stability under harsh conditions. Considering their structural diversity and similarity to pharmaceutical building blocks, 10 aliphatic amines were selected for enzymatic production8,18. This includes two amines with cycloalkyl substituents (C1 and C2 in Table 1).
To compare the performance of PjAT-R6 to other well-characterized (S)-selective TAs, we included the enzymes from Chromobacterium violaceum (CvTA) and Vibrio fluvialis (VfTA). Through a systematic analysis of the substrate spectrum of various transaminases reported in different papers Calvelage et al.25 concluded that CvTA and VfTA have advantages in converting straight-chain amines over other transaminases. The relative performance of PjTA-R6 in
comparison to CvTA and VfTA may help to evaluate the enzyme in terms of its significance within the enzymatic toolbox. Under the employed conditions, the yields of aliphatic amines obtained with PjTA-R6 were higher than with CvTA or VfTA, and the enantiopurity of the produced amines was also higher.
Aiming to understand the structural basis for the observed reactivities of PjTA-R6 and VfTA, we performed docking calculations of the external aldimine complexes of the tested substrates. For both enzymes, the interface energies of the external aldimine complexes correlated well with experimental reaction yields, indicating that biocatalytic scope can be accessed
computationally for further redesign attempts. The docking complexes hinted to pivotal structural differences in the way PjTA-R6 and VfTA accommodate bulky or alkyl chains in the large substrate binding pocket.
Results and discussion
Asymmetric synthesis of aliphatic amines
To investigate the performance of PjTA-R6 in the asymmetric synthesis of aliphatic amines and compare the results with CvTA and VfTA, amination reactions were conducted using the three enzymes and a series of keto substrates (Table 1). For these amination reactions, we adopted conditions with an 50-fold excess of isopropylamine (IPA) as the amino donor (Scheme 1). This amino donor is relatively cheap7 and highly water soluble, making its use in enzymatic aminations economically attractive. Moreover, IPA is transformed to acetone, whose volatility may simplify the work-up procedure26. The reaction temperature was 56 °C for PjTA-R6, given its high
thermostability,and 37 °C for CvTA and VfTA. Protein concentrations were the same for all three enzymes. Noticeably, after 20 h of reaction, an enzyme precipitate was observed in the vials
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containing CvTA and VfTA but not in the reaction mixtures containing PjTA-R6. This agreed with our expectation that PjTA-R6 would be more robust than the other two transaminases at high reaction temperature with excess amino donor present.
Table 1. Analytical yields and enantiomeric excesses for the production of A1-A5, B1-B3, and C1-C2 using PjTA-R6, CvTA, and VfTA.
Amine target code
Amine structure
PjTA-R6 CvTA VfTA
Yield (%) ee (%) Yield (%) ee (%) Yield (%) ee (%) A1 70 94 40 86 5 n.d. A2 80 98 34 91 12 41 A3 51 >99 23 91 14 88 A4 51 >99 44 90 41 92 A5 42 >99 46 88 35 94 B1 95 >99 3 n.d. 1 n.d. B2 81 >99 3 n.d. 2 n.d. B3 48 >99 <1 n.d. 2 n.d. C1 42 >99 1 n.d. <1 n.d. C2 63 >99 <1 n.d. 1 n.d.
1 n.d.: not determined; ee: enantiomeric excess. 2 The yields obtained with PjTA-R6 are the
averages of duplicates that differed less than 10%. 3 Reaction conditions: 1 mL volume, 20 mM
ketone substrate, 1 M isopropylamine, 0.5 mM PLP, 1 mg/mL purified transaminase, 2% DMSO (20% for A5), 20 h, 56 °C for PjTA-R6, 37 °C for CvTA and VfTA.
The asymmetric synthesis of aliphatic amines was successful as all ketone substrates were converted by PjTA-R6 and yielded predominantly (S)-amines with all yields exceeding 40% (Table 1). Whereas equilibrium may have been approached in some reactions (e.g. B1 with PjTA), the lower yields with most other substrates and especially in reactions catalyzed by CvTA and VfTA indicate that differences in yields were mainly determined by kinetics. In particular, amines
A1 and A2 and branched amines B1 and B2 were well synthesized by PjTA-R6 . Noticeably, using
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C1, which has a cyclopropyl group. This suggested that general bulkiness of the larger substrate is
not limiting conversion. Chiral analysis of the amines formed by PjTA-R6 confirmed the expected (S)-enantioenrichment, and all amines were produced at high ee. The enantiopurities of the
products indicated a dependence on the difference in size between the large (RL) and small (RS = -CH3) side substituents on the carbonyl carbon of the substrates. A1 (RL = -CH2CH3) and A2 (RL = -CH2CH2CH3) were produced with 94% and 98% ee, respectively, while all the other amines were produced with >99% ee.
Under the employed conditions, the yields of straight-chain amines (A1-A5) in reactions using PjTA-R6 were generally better than those with CvTA and VfTA. For the production of A5, PjTA-R6 and CvTA displayed comparable results (Table 1). The yields found with PjTA-R6 were better in case of straight-chain aliphatic amines with shorter chains (A1-A2) than with amines with larger groups (A3-A5). In contrast, VfTA showed an improving trend in the yields with an
increasing chain length of the target amines. A previous study on the activity of VfTA toward a set of alkyl substrates also showed that the longer the side chain, the higher the activity27. For CvTA, no such trend was found. For the production of A1-A3, PjTA-R6 generated higher yields by 28% or more than CvTA while VfTA gave the lowest yields. The yields of A4 and A5 obtained with the three enzymes differed by less than 11%. The general advantage of asymmetric synthesis using PjTA-R6 was also clear from its better enantioselectivity. Whereas CvTA and VfTA produced linear aliphatic amines with ee < 94%, all amines formed by PjAT-R6 had better than 94% ee.
For amines with branched carbon chains (B1-B3) and cycloalkene groups (C1-C2), PjTA-R6 generated high product yields, similar to those of amines with linear substituents (Table 1). In particular, B1 was produced with 95% yield, which was the highest yield obtained with PjTA-R6. The advantage of PjTA-R6, compared with CvTA and VfTA, were again apparent from the higher yields. The conversion of amines with non-linear substituents by CvTA and VfTA was not
successful. This suggests that the presence of a non-linear large rest group in the substrate prevents proper accommodation of the ketone or subsequent reaction intermediates in these enzymes. Overall, PjTA-R6 had a better performance in yield and an excellent enantioselectivity, which makes PjTA-R6 a promising candidate for the asymmetric amination of aliphatic ketones. The difference between the three enzymes for the better converted substrates can be due to kinetic and stability effects. Differences observed for the same enzyme should be attributed to kinetic
differences, unless conversion is very high when equilibrium may be approached (Table 1, PjTA-R6 with A2, B1). For the branched and cyclic substrates (B1-B3, C1, C2) which are well
converted by PjTA-R6 but poorly converted by CvTA and VfTA, low intrinsic catalytic activity of the latter two enzymes must be the cause of the observed differences.
Role of switching arginine
As mentioned, the native substrate of PjTA is the caprolactam biodegradation intermediate 6-aminohexanoic acid (6-AHA)22. The crystal structure of PjTA complexed with the external aldimine formed from this substrate unveils that the carboxylate group forms a salt bridge with a
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conserved arginine residue (Arg417) in the large pocket, probably contributing to substrate
binding23. The arginine side chain switches towards a more outward position upon 6-AHA binding. Arg417 and most surrounding residues were kept during the thermostability engineering that yielded PjTA-R624. During the redesign of other class III (S)-selective transaminases the switching arginine is mostly preserved since amino donors or acceptors often feature a carboxylate
functionality27−29. However, in VfTA, Arg415 has been mutated to open up more space in the large pocket27,29,30. For the synthesis of aliphatic alkylamines with IPA as amino donor neither Arg415 nor Arg417 of VfTA and PjAT-R6, respectively, are required and the iminium group could even produce repulsive interactions.
To examine whether Arg417 still has a role in substrate binding when using isopropylamine as amino donor and an amino acceptor substrate lacking a carboxylate
functionality, the R417L mutant of PjTA-R6 was prepared and tested in the same reaction scheme as for PjTA-R6. Thermal shift fluorescence assays showed that the thermostability of the R6-R417L enzyme was similar to that of the parent PjTA-R6 (∆Tmapp < 1 °C). The yields of the enzymatic amination using the mutant R417L remained at the same level as with PjTA-R6. Yields of A1 and A2 obtained with R6-R417L (60% and 69%, respectively) were only slightly lower than that with PjTA-R6 (70% and 80%). As for A3-A5, the yields obtained with R6-R417L (54, 50 and 47%) were very close to the yields with PjTA-R6. Apparently, the positive charge of the arginine does not hinder activity with the apolar aliphatic acceptors tested here. A possible explanation is conformational flexibility of the aliphatic group of the substrates or of the Arg417, which may avoid close contact with the positively charged iminium function, unlike what is observed in the 6-AHA-bound PjTA structure23. We anticipate that Arg417 can be replaced for future engineering needs, e.g. aimed at expanding the binding site for more bulky substrates.
Structure-activity relationship obtained by docking
Accommodation of the external aldimine docking complexes formed with different substrates by PjTA-R6 and VfTA were examined to find a structural explanation for the observed activity profiles (Table 1). The substrate-binding pockets of these TAs are positioned at the dimeric interface and consist of residues from both subunits. Because the small substituent of the tested molecules was always a methyl group, the structure of the binding pocket that accommodates the large substituent is crucial for differences in substrate specificity. The large binding pocket accommodates the carboxyalkyl-moiety or large substituent of 6-AHA in PjTA, and the phenyl substituent of (S)-1-phenylethylamine in VfTA. Structural alignment of PjTA-R6 with CvTA (40% sequence identity, RMSD 1.28 Å) and with VfTA (41% sequence identity, RMSD 1.20 Å)
indicates a high overall similarity, nonetheless with local differences in the large binding pockets (Figure 1). The side chains of Leu57, Tyr151, Arg417, Met419, Ala230, Trp58, Val260, and Ile261 form a largely non-polar binding pocket in PjTA-R6. The corresponding pockets of CvTA and VfTA are also hydrophobic. To examine the features that potentially led to variations in substrate specificities and confer PjTA-R6 with high reactivity toward a variety of bulky amines,
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structures of enzyme complexed with of the product-derived external aldimines were generated by Rosetta docking.
Figure 1. Schematic representation of the binding pocket of PjTA-R6 bearing the external aldimine intermediate [PLP-Schiff base with (S)-1-phenylethylamine] and comparison of surrounding residues to
CvTA and VfTA. The residues in the large binding pocket are in blue. The residues colored red are in the
small binding pocket.
The Rosetta interface energies obtained from docking the external aldimine complexes of the ten different substrates showed a clear correlation with the experimental data observed for both VfTA and PjTA-R6 (Figure 2). Nevertheless, outliers are present in both plots. The most prominent outlier in the VfTA dataset is compound B3, which exhibits a higher docking score than would be expected based on the experimental yield. B3 is also one of the three outliers in PjTA-R6, along with A4 and A5. The latter two are the only two compounds in the PjTA-R6 dataset where the switching arginine Arg417 in the docking complex is kept in a crystal-like “out” conformation (discussed later), whereas all the other compounds (A1-A3, B1-B3, C1-C2) show Arg417 in an “in” conformation. The lower than expected yield for A5 with PjTA-R6 may also be due to the higher concentration of DMSO used to dissolve this substrate. The correlation between interface energies and experimental yields suggests that structural differences in the binding pockets can account for most of the observed differences in activity within a set of substrates tested with the same enzyme. Since for each enzyme at least one substrate was very well converted, low yields for several substrates, such as B1-C2 for CvTA and VfTA, must be due to kinetic causes rather than to rapid enzyme inactivation. However, differences in obtained yields between different enzymes may be related both to kinetic and stability differences under the reaction conditions.
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Figure 2. Interface energies of external aldimine complexes show a correlation with the observed activities. A) PjTA-R6. B) VfTA. Outliers (red dots) were not considered for the linear regression (dotted line). For
PjTA-R6, this means the regression was made using compounds A1, A2, A3, B1, B2, C1, and C2. In the
case of VfTA, B1, B2, C1, and C2 were not considered for the linear regression since product was hardly detectable (<2%). The Rosetta Interface Energy correctly predicts that B2, C1, and C2 will have very low yields, hence these compounds are not outliers.
The activity data suggest that PjTA-R6 can accommodate bulkier substituents than VfTA (Table 1). The docking simulations revealed that the conformational freedom of the switching arginine plays a large role in the ability of PjTA-R6 to accommodate bulky substrates. In both VfTA and PjTA-R6 the switching arginine (Arg415 and Arg417, respectively) can adopt one of two conformations that allow the binding of hydrophobic substituents in the large pocket (Figure 3).
Figure 3. Docked external aldimine structures of 3,3-dimethyl-2-butylamine B2, representative of a products with bulky substituents. A) In PjTA-R6, the tert-butyl substituent can be easily accommodated in the large pocket by a 40° rotation of Trp58 (yellow arrow). Arg417 does not induce protein-ligand clashes in neither the “in” nor the “out” orientation. B) In VfTA, both the “in” and the “out” conformations of Arg415 prevent a rotation of Trp57 necessary to accommodate the tert-butyl substituent. In both panels, protein structures are shown in licorice representation, and the crystal structure conformations of residues Arg417/Arg415 and Trp58/Trp57 are colored magenta (PDBs 4Q3H for VfTA, and 6TB1 for PjTA-R6). The external aldimine structure of B2 is shown in CPK representation. Figures were rendered using VMD.
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We labeled these Arg conformations as “in” and “out” to indicate whether the guanidium group is pointing inward (towards) the PLP or outward. The “in” and “out” arginine conformations in VfTA and PjTA-R6, respectively, have been crystallized. In the known VfTA structures (PDB codes: 4E3Q, 4E3R, 5ZTX, 3NUI), Arg415 is stabilized in the inward conformation by a hydrogen bond with Trp57. As such, the “in” conformation appears to be the most stable for the PLP-bound enzyme and the apo-enzyme. On the other hand, the “out” arginine conformation prevails in the known PjTA structures (PDB codes: 6TB1, 6TB0, 6G4B, 6G4C, 6G4D, 6G4E, 6G4F), where Arg417 is stabilized by electrostatic interactions, either with Ser87 in the wild-type PjTA or with Asn87 in PjTA-R6. In PjTA-R6, Ser87 has been mutated to Asn to improve the enzyme’s thermostability15,16. The natural substrate of PjTA is 6-AHA, where the role of the switching arginine is to form a salt bridge with the carboxyl group of the substrate (code: 6G4E). PjTA-R6 and VfTA exhibit structurally different “in” and “out” Arg conformations. The outward
conformation in VfTA places the Arg415 side chain in a sandwich-like configuration between Trp57 and Asn53, reducing the ability of Trp57 to freely rotate in response to the presence of a bulky substrate. On the other hand, the out conformation of PjTA-R6 moves Arg417 away from the binding pocket, locking Arg417 with Asn87 via a polar interaction, thereby both opening up more space in the large pocket and allowing free rotation of Trp58. The inward conformations of Arg415/Arg417 in VfTA and PjTA-R6 can also account for the difference in reactivity toward bulky substrates. In the “in” conformation, the switching arginine of PjTA-R6 makes hydrogen bonding contacts with the backbone of Ala230, leaving enough space in the large pocket for bulky substituents. In contrast, VfTA locks Arg415 with the indole amino group of Trp57 via hydrogen-bonding interactions, offering less space in the large pocket. Mutations of Trp57 have been carried out, yielding good reactivities toward both alkyl and aromatic substrates20. Arg415 from VfTA cannot reach the backbone of the equivalent Ala228 due to the presence of Leu417, which corresponds to Met419 in PjTA-R6. These differences in both the “in” and “out” conformations can explain the distinct catalytic performance of the two enzymes towards bulky substrates.
An additional difference between PjTA-R6 and VfTA is the effect of increasing the chain length of the alkyl substituent on activity (i.e. substrates A1-A5). The experimental data show that longer alkyl substituents negatively impact conversion by PjTA-R6 (Table 1). On the contrary, VfTA (and CvTA) shows better activities with the longer alkyl substituents, as also found by Cho et al. in a study aimed at redesigning the substrate specificity of VfTA27. Rosetta-generated docking models of the external aldimine intermediates of A1-A5 indicate that VfTA accommodates the increasingly longer alkyl chain in the large pocket by pointing the terminal methyl group of the alkyl chain toward the entry tunnel (Figure 4B). In the case of PjTA-R6, the equivalent space is partially blocked by Arg417 in both the “in” and “out” conformations. The terminal methyl group of the alkyl chain of A4 and A5 is still perfectly able to fit in the large pocket of PjTA-R6, albeit less favorably (Figure 4A) than compounds with shorter alkyl substituents (i.e. A1-A3). The negative effect of Arg417 on the activity of PjTA-R6 is minor, as demonstrated by the fact that mutation R417L offers only a marginal improvement in the yields of amines A4 and A5.
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Furthermore, the large pocket of PjTA-R6 contains more polar residues than VfTA, thereby offering a less suitable environment to accommodate the lengthening alkyl chain of the A1-A5 series. All in all, while the catalytic activity of PjTA-R6 decreases for long alkyl chains, PjTA-R6 still gave higher yields and higher enantioselectivities than VfTA, making PjTA-R6 valuable for synthesis of enantiopure alkyl amines.
Figure 4. Docking structures of the external aldimine intermediates of amines A1 – A5. A). PjTA-R6 B)
VfTA. The “out” conformation of the switching arginine is preferred for longer substituents (A4 and A5),
whereas the shorter alkyl substituents (A1 to A3) show no preference for either conformation. The large binding site of R6 is more hydrophobic than the binding site of VfTA (N87, Y20, and F86 for PjTA-R6, and F86, F19, and F85 for VfTA, respectively). The superimposed external aldimine structures of A1 – A5 are shown in CPK representation. The “in” conformation of Arg417/Arg415 is colored magenta.
Materials and methods
Materials
Isopropylamine, amines (A1-A5, B1-B3, C1-C2), and the corresponding ketones were purchased from Sigma-Aldrich or Acros Organics. PLP was purchased from Fisher Scientific. Other
chemicals were from the same suppliers or Merck Millipore.
Enzyme expression and purification
The design, isolation and properties of the PjTA-R6 mutant was described24. The PjTA-R6-R417L mutant was created by QuikChange site-directed mutagenesis. Primers were designed by the QuikChange Primer Design Program of Agilent Technologies. The mutations were verified by DNA sequencing (Eurofins Genomics, Germany). After sequencing, the mutated plasmids were transformed into E. coli BL21(DE3) for expression. The expression and purification of PjTA-R6, PjTA-R6-R417L, CvTA and VfTA was performed as previously described. Specific activities of purified enzymes in the deamination of (S)-1-phenylethylamine were 47 U/mg (at 70C,
spectrophotometric acetophenone production assay24), or 1.6 and 1.0 U/mg (at 30C, coupled enzyme assay using pyruvate as amino acceptor23) for PjTA, CvTA and VfTA, respectively.
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Thermal shift assay
Apparent melting temperatures (Tmapp) were determined by the fluorescence-based thermofluor assay31. Specifically, samples of 5 µL 50-fold diluted SYPRO orange and 20 µL 0.5 mg/ml
enzyme solution were added to an IQ 96-well PCR plate (Bio-Rad, USA) and mixed thoroughly in the wells. The plates were sealed with Microseal B adhesive sealer (Bio-Rad, USA) and heated from 20 to 99 °C in a MyiQ real-time PCR machine (Bio-Rad, USA) with a linear gradient of increasing temperature (1 °C/min). The temperature at the maximum rate of fluorescence change (dRFU/dT) was taken as Tmapp32.
Amination reactions and product derivatization
Enzyme reactions were carried out in a 1 mL reaction system containing 50 mM potassium phosphate, pH 8.0, with 20 mM ketone substrate, 1 M isopropylamine, 0.5 mM PLP, 1 mg/mL enzyme, and 2% DMSO (20% for A5). Reactions were started by addition of substrate and carried out at 56 °C for PjTA-R6 and R417L and 37 °C for CvTA and VfTA over 20 h. Reactions were stopped by adding 200 µL 10 M NaOH, after which 100 µL samples were taken from the basified solutions and extracted once with 300 µL EtOAc.
For derivatization, the organic layer was supplied with 300 µL acetic anhydride and 4-dimethylaminopyridine (DMAP)1, and the solution was shaken at room temperature and 450 rpm for 1 h. Following washing with 500 µL water, the organic layer containing the derivatized product was collected and dried over magnesium sulfate.
Quantification and optical purity
The derivatized products were analyzed by gas chromatography with flame ionization detection on a heptakis-(2,6-di-O-methyl-3-O-pentyl)-β-cyclodextrin column using He at a flow rate of 1 mL/min. The following settings applied to the analysis of all products and their corresponding commercial references: temperature program: 80 °C for 6.5 min, 10 °C/min to 160 °C, 20 °C/min to 200 °C for 10 min; total run time: 26.5 min; inlet temperature: 220 °C; injection volume: 1 µL. To quantify the product, the peaks of the enantiomers were summed up and compared with a commercial amine reference (see Supplementary Materials). To analyze the optical purity of the products, the peaks of different enantiomers were mostly identified with commercial chiral amine references (see Supplementary Materials). The ee was calculated from the peak areas of the (S)- and (R)-amines.
Computational modeling materials
The coordinates for the (S)-amino products of the tested compounds (A1–A5, B1–B3, C1–C2) were generated in Avogadro (Hanwell et al., 2012; https://avogadro.cc/)26 from their respective SMILES string, and optimized by 500 steps of steepest descent energy minimization in the
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were created by superimposing the amino group of the abovementioned (S)-amino products to the amino group of the PLP cofactor, whose coordinates were obtained from the crystal structure (PDB 4E3Q). After superimposition, one of the nitrogen atoms was deleted (the cofactor’s), and a
covalent bond was added between C4’ and the remaining nitrogen atom (the amino substrate’s), to form the corresponding Schiff base. A rotamer library for each ligand was generated in
YASARA33. For each external aldimine structure, 1,000 rotamers were generated by random perturbation of the heavy-atom dihedrals (SampleDih YASARA routine), while maintaining the cofactor atoms frozen. Pairwise RMSD between all rotamers within each library was used for selecting unique rotamers (cutoff < 0.05 Å), resulting in 200–300 unique rotamers for each ligand.
Structure coordinates for PjTA-R624 and VfTA30 were obtained from the PDB databank (6TB1 and 4E3Q, respectively). The crystal structures were prepared for docking by first implementing the CleanObj YASARA routine, which added explicit hydrogens to the structure, and converted Se-Met residues into canonical Met. Protonation states of residues Asp, Glu, His, and Lys were calculated by an automatic YASARA routine at pH 733. The resulting scaffold structure was stripped of any non-protein residues.
The scaffold protein structure was relaxed by the Rosetta (v2020.11) Relax34 algorithm with the following commandline arguments: ignore_unrecognized_res,
-relax:constrain_relax_to_start_coords, -relax:coord_constrain_sidechains, -relax:ramp_constraints false, -use_input_sc, -correct, -no_his_his_pairE, -no_optH false, -flip_HNQ. Binding poses of external aldimine complexes were generated using the Rosetta Enzyme Design application35. One randomly selected rotamer from each conformer library was overlapped to the binding site (subunit A) of PjTA-R6 or VfTA by RMSD alignment to the cofactor heavy atoms. The resulting structure served as input for the docking calculations. During the docking simulations, residues with Cα closer than 10 Å from any ligand heavy atom were allowed to repack. For PjTA-R6, residues 20, 87, 59, 118, 119, 151, 152, 225, 259, 287, 292, 324 were set to NATRO (kept fixed). For VfTA, residues 19, 53, 115, 118, 121, 150, 151, 256, 258, 322 were set to NATRO. No constraints were used in the production runs. Additionally, the following command-line arguments were used for the Rosetta docking: enzdes, cst_predock, cst_design, cut1 0.0 cut2 0.0, cut3 8.0, cut4 10.0, cst_min, chi_min, bb_min, packing::use_input_sc, packing::soft_rep_design,
-design_min_cycles 3, -ex1:level 4, -ex1:level 4, -ex1aro:level 4, -ex2aro:level 4. A total of 200 docking complexes were generated for each ligand, and the average interface energies computed and compared against the experimental yield. Figures were generated using VMD
(http://www.ks.uiuc.edu/Research/vmd/).
Conclusions
PjTA-R6 was capable of converting various aliphatic ketones into the corresponding amines with good activity, also when compared to CvTA and VfTA. The scope of aliphatic amines that can be asymmetrically synthesized spans from amines with linear (A1-A5) or branched substituents
(B1-109
B3) to lesser studied amines with a cycloalkyl moiety (C1-C2). The enantioselectivity of PjTA-R6
was ideal for most cases, which plays in favor of using this enzyme for chiral amine production. PjTA-R6 catalyzed amination at a relatively high temperature (56 °C). Its robustness allows use of the enzyme under beneficial conditions for enzymatic amination, e.g. high temperature, higher amino donor concentration and presence of cosolvent for better substrate and product solubility. The mutant Arg417L showed a comparable performance to PjTA-R6, indicating that the switching arginine Arg417 is replaceable when a carboxylate functionality is absent from the keto substrate and the amino donor.
The Interface Energies obtained with Rosetta docking simulations of the external aldimine reaction intermediates showed a clear correlation with yield in amine synthesis, both in case of PjTA-R6 and VfTA. Further comparison of the docked structures unveiled differences in the conformations of the switching arginine that interacts with the native substrates of PjTA-R6 and VfTA. In VfTA the conformational freedom of the switching arginine is more restricted than in PjTA-R6, and does not allow VfTA to accommodate bulky substrates. Overall, the binding site of PjTA-R6 is better at adapting to fit bulkier substituents than the related enzyme VfTA. The observation that the relative reactivity of PjTA-R6 can be rationalized by docking simulations and energy calculations suggests that these computational tools can support the design of variants with expanded or shifted substrate spectrum, which is important for furthering applicability in green chemistry.
Author Contributions
QM supervised and supported the experimental work and wrote the corresponding part of the manuscript. LD performed the experimental work and wrote the manuscript. CRP performed the docking analysis and wrote the corresponding part of the manuscript. HJW and SJM revised the manuscript. DBJ revised the manuscript and supervised the project.
110
Supporting information
Reaction scheme and derivatization
111
Assignment of absolute configuration
The absolute configuration was assigned comparing the elution order with amine references prepared with commercially available racemic amines and/or (S)- or (R)-amines. The amine references were prepared in the phosphate buffer (20mM amine, 1 M isopropylamine, 0.5 mM PLP, 2% DMSO for A1-A4, B1-B3, C1, and C2, 20% DMSO for A5) resembling the reaction mixture of 100% yield. The amine references of different concentrations would be derivatized and analyzed according to the derivatization method as described in Materials and methods.
To serve as a representative procedure, 20 mM, 2 mM, and 0.2 mM A3 solutions were prepared in phosphate buffer as in reaction mixture without the enzyme (1 M isopropylamine, 0.5 mM PLP, 2% DMSO). In solutions of different concentrations, the ratios of (S)- and
(R)-enantiomers were kept at 3:1. After derivatization and GC analysis, the retention times of two enantiomers can be identified from the chromatograms (Figure S1).
112
Yield and enantiomeric excess
The yield of all product was determined by comparing to commercial amine references resembling 100% yield (20 mM). As for R6, the reaction was performed in duplicates and the yields were shown as averages that differed from each other less than 10%. The enantiomeric excess was determined after comparing the peaks of the derivatized (S)- and (R)-enantiomers. The retention time are listed in Table S1. The representative chromatograms of the product amine (A1-A5, B1-B3, C1, and C2) from the transamination using PjTA-R6 are shown below.
Table S1. The retention times of the derivatized products. Amine target
code
Retention time of derivatized amine* (min) (S)-enantiomer (R)-enantiomer A1 12.2 12.3 A2 13.2 13.4 A3 14.7 14.9 A4 15.8 16.0 A5 16.7 16.8 B1 12.9 13.0 B2 13.3 13.5 B3 13.7 13.9 C1 13.9 14.2 C2 17.5 17.6
*: The retention time of derivatized amines shifted over the prolonged use of the chiral column. Assignment of absolute configuration was always confirmed using commercial amine references.
113
Figure S2. Representative chromatogram of PjTA-R6 catalyzed transamination of 2-butanone yielding sec-butylamine (A1) after product derivatization. Left peak corresponds to the derivatized product of (R)-A1, while the right corresponds to the derivatized product of (S)-A1.
Figure S3. Representative chromatogram of PjTA-R6 catalyzed transamination of 2-pentanone yielding 2-aminopentane (A2) after product derivatization. Left peak corresponds to the
114
Figure S4. Representative chromatogram of PjTA-R6 catalyzed transamination of 2-hexanone yielding 2-aminohexane (A3) after product derivatization. Peak corresponds to the derivatized product of (R)-A3, while the derivatized product of (S)-A3 is below detection limit.
Figure S5. Representative chromatogram of PjTA-R6 catalyzed transamination of 2-heptanone yielding 2-aminoheptane (A4) after product derivatization. Peak corresponds to the derivatized product of (R)-A4, while the derivatized product of (S)-A4 is below detection limit.
115
Figure S6. Representative chromatogram of PjTA-R6 catalyzed transamination of 2-octanone yielding 2-aminooctane (A5) after product derivatization. Peak corresponds to the derivatized product of (R)-A5, while the derivatized product of (S)-A5 is below detection limit.
Figure S7. Representative chromatogram of PjTA-R6 catalyzed transamination of 3-methyl-2-butanone yielding 3-methyl-2-butanamine (B1) after product derivatization. Peak corresponds to the derivatized product of (R)-B1, while the derivatized product of (S)-B1 is below detection limit.
116
Figure S8. Representative chromatogram of PjTA-R6 catalyzed transamination of 3,3-dimethyl-2-butanone yielding 3,3-dimethyl-2-butanamine (B2) after product derivatization. Peak
corresponds to the derivatized product of (R)-B2, while the derivatized product of (S)-B2 is below detection limit.
Figure S9. Representative chromatogram of PjTA-R6 catalyzed transamination of 4-methyl-2-pentanone yielding 4-methyl-2-pentanamine (B3) after product derivatization. Peak corresponds to the derivatized product of (R)-B3, while the derivatized product of (S)-B3 is below detection limit.
117
Figure S10. Representative chromatogram of PjTA-R6 catalyzed transamination of
1-cyclopropylethanone yielding 1-cyclopropylethanamine (C1) after product derivatization. Peak corresponds to the derivatized product of (R)-C1, while the derivatized product of (S)-C1 is below detection limit.
Figure S11. Representative chromatogram of PjTA-R6 catalyzed transamination of 1-cyclohexylethanone yielding 1-cyclohexylethanamine (C2) after product derivatization. Peak corresponds to the derivatized product of (R)-C2, while the derivatized product of (S)-C2 is below detection limit.
118
Figure S12. SDS-PAGE analysis of purified CvTA (51.2 kDa), VfTA (52.3 kDa), and PjTA-R6 (49.9 kDa).
119
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